Maintenance of the translational reading frame is a fundamentally important feature of protein synthesis. There is clear evidence that ribosomes contain an "exit" or "E" site for deacylated tRNA, and that tRNA pass through the E site after completing their translational role. But except for transient interactions with the exiting tRNA, no other clear function has been identified for the E site. However, recent evidence leads to the hypothesis that deacylated tRNA in the ribosomal E site helps prevent frameshifting. We propose four experimental tests of this hypothesis. We will determine whether the E site is holds tRNA until an aminoacyl- tRNA is selected at the A site. If so, then the E site tRNA might have an important functional role in translation of the A site. If not, then the E site is probably just an exit path. We will also determine whether message nucleotides and tRNA sequences in the E site are associated with increased or decreased frameshifting at the RF2 programmed frameshift site. Preliminary work shows that the E site triplet is important, although the mechanisms are not yet clear. Proposed work will define its role. Finally, we will determine whether an E site tRNA establish the reading frame on ribosomes in vitro. [unreadable] [unreadable] [unreadable]